Human tryptase, a tetrameric proteinase expressed by mast cells, is virtually unique among the serine proteinases as it is not inhibited by any proteinaceous inhibitor tested so far. We have now isolated, sequenced, and characterized an inhibitor of human tryptase from the medical leech Hirudo medicinalis. LDTI (Leech-Derived Tryptase Inhibitor) was purified to apparent homogeneity by cation exchange and affinity chromatography. Amino acid sequencing of the protein consisting of 46 residues (M(r) 4738) revealed a high degree of similarity to the non-classical Kazal-type inhibitors bdellin B-3 and rhodniin, inhibitors isolated from the medical leech and the insect Rhodnius prolixus, respectively. LDTI is a tight-binding and relatively specific inhibitor of human tryptase; it inhibits only trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) with similar affinities. Inhibition studies using small chromogenic substrates revealed that LDTI inhibits the amidolytic activity of tryptase by approximately 50%, suggesting that most likely due to steric hindrance LDTI binds to and inhibits only 2 of 4 active sites of tryptase. LDTI appears useful as a prototype of inhibitors of human tryptase and as a pharmacological tool for the investigation of the role of tryptase in health and disease.

Antistasin, a potent inhibitor of the blood coagulation factor Xa, is the prototype of a novel family of serine-proteinase inhibitors. We have now isolated, sequenced and characterized an antistasin-type inhibitor from the medical leech Hirudo medicinalis. Hirustasin (Hirudo antistasin) was purified to apparent homogeneity by cation-exchange and affinity chromatography. Amino acid sequencing of the 55 amino acid protein (MI 5866) revealed that hirustasin is the only antistasintype protein known to consist of one domain only; 27% and 32% sequence identity was found to the first and second domains of antistasin, respectively, and a nearly exact conservation of the spacing of the ten cysteine residues. Hirustasin is the first inhibitor of tissue kallikrein identified in leeches, and is also a tight-binding inhibitor of trypsin, chymotrypsin and neutrophil cathepsin G. However, despite the high similarity to antistasin, particularly in the vicinity of the putative reactivesite peptide bond, hirustasin neither inhibits blood coagulation in vitro nor amidolytic activity of isolated factor Xa. Thus, structural elements other than the reactive site sequence significantly influence the specificity of antistasin-type proteinase inhibitors.Antistasin, a polypeptide of 119 amino acids, is a cysteine-rich serine-proteinase inhibitor originally isolated from the salivary glands of the Mexican leech Haementeria ofj'icinalis [l]. Considerable interest has been focused on this protein as it is a highly selective, tight-binding inhibitor of the blood coagulation factor Xa and thus a potent anticoagulant in vitro and in vivo [2-61. In addition, antistasin seems to have marked antimetastatic properties El].Amino acid sequence analysis revealed that antistasin contains two homologous domains ; no sequence similarity was found to other known proteins suggesting that antistasin is the prototype of a new family of serine-proteinase inhibitors [7]. Meanwhile, another protein has been isolated from the giant Amazonian leech Haementeria ghilianii, ghilanten,